Horizontal electrophoresis or isoelectric focusing apparatus and method of using same

ABSTRACT

Electrophoresis or isoelectric focusing of a starting species, in free solution, is performed in an elongated horizontal vessel, following which the vessel is turned about its horizontal longitudinal axis in order to move the separated fractions into separate collecting compartments contained within the vessel.

This is a division of application Ser. No. 29,063 filed Apr. 11, 1979now U.S. Pat. No. 4,234,404.

This invention relates to methods for performing horizontalelectrophoresis or horizontal isoelectric focusing of starting speciesin free solution, in which the separated fractions are collectedseparately in compartments in the same vessel in which theelectrophoresis or isoelectric focusing is performed, and an apparatusfor use in performing those methods.

Electrophoresis and isoelectric focusing are well-known fractionationmethods for separating molecules, bioparticles and macromolecules. Inelectrophoresis, particles or molecules having a net electric chargemigrate electrophoretically, under the influence of an electric field,typically at a constant pH of, for example, about 8.6, at various ratesdepending on their electrophoretic mobility, toward the positive ornegative electrode. In isoelectric focusing, amphoteric molecules andbioparticles, i.e., species capable of exhibiting positive, negative orzero electric charge as a function of pH, migrate electrophoretically,under the influence of an electric field and a pH gradient, until theyare condensed or focused at positions that correspond to theirrespective isoelectric points whereat their net charge approaches zero.Although the principles of fractionation are different in the twomethods, the two methods are closely related fractionation methods andsimilar procedures and apparatus can be used in the two methods. Aprincipal operational difference in the two methods is that inisoelectric focusing a carrier ampholyte liquid is used, whereas inelectrophoresis an electrically conductive electrophoresis medium isused.

This invention is useful for both electrophoresis and isoelectricfocusing. For purposes of simplifying the following description, theterm "electrophoresis" will be principally used in the followingdescription, but it will be understood that the term refers to bothelectrophoresis and isoelectric focusing, unless the contextspecifically requires otherwise.

Further, in the following description, the term "starting species" meansthe starting material that is to be subjected to electrophoresis inorder to effect separation of the components thereof. For example, thestarting species can be a mixture of lymphocytes or a mixture of serumproteins which is to be subjected to electrophoresis in order to resolvesame into fractions of more closely homogeneous characteristics.Moreover, the term "free solution" refers to the fact that the startingspecies is carried in a liquid carrier medium so as to be freely movabletherein. The starting species can be dissolved or suspended in theliquid carrier.

A wide variety of satisfactory preparative electrophoresis methods andapparatus are known in the art. However, the prior art apparatuses whichare useful for obtaining a large number of separate fractions of thestarting species are quite complex and expensive, and the methods ofusing them are time-consuming and technically involved. There is a needfor a simpler, less expensive and easier-to-use electrophoresis methodfor separating a starting species into a large number of fractions andrecovering those fractions.

It is an object of this invention to provide an improved electrophoresismethod in which the starting species is placed in an elongatedhorizontal vessel and then is subjected to electrophoresis therein toseparate the starting species into fractions which are displacedhorizontally along the lengthwise extent of the horizontal vessel,following which the vessel is turned about its horizontal longitudinalaxis so that the separate fractions are moved into and are separatelycollected in different mutually isolated compartments in the vesselwhereby the fractions can be recovered separately from one another withminimal cross-contamination of the fractions.

It is a further object of this invention to provide an improvedelectrophoresis method which ca be used to separate human peripheralblood lymphocytes by electrophoresis.

It is a further object of this invention to provide an improved method,as aforesaid, in which amphoteric macromolecules or cells can beseparated into fractions by isoelectric focusing.

It is another object of this invention to provide an improved method, asaforesaid, in which the starting species, in free solution, migrateelectrophoretically in a horizontal direction in the vessel, under theinfluence of an electric field and under conditions which minimize therisk of damage to components of the starting species.

It is a further object of this invention to provide an improved method,as aforesaid, which is simple and easy to use, which is rapid inoperation, and which does not require the use of a density gradientmaterial or separate collecting vessels for receiving the separatedfractions.

Additional objects and advantages of the invention will become apparentupon reading of the following detailed description and inspection of theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a front view, partially broken away, of a horizontalelectrophoresis vessel according to the invention.

FIG. 2 is an end view of the electrophoresis vessel of FIG. 1.

FIG. 3 is a sectional view, taken along the line III--III of FIG. 1,showing the cross section of the electrophoresis vessel, shown in thenormal position thereof while the electrophoresis operation is beingcarried out.

FIG. 4 is a view similar to FIG. 3, but in which the electrophoresisvessel has been turned through an arc of about 120° about its horizontallongitudinal axis, after completion of the electrophoresis operation, sothat the separated fractions are received in the compartments in thevessel.

FIG. 5 is a sectional view taken along the line V--V of FIG. 3.

FIG. 6 is a sectional view taken along the line VI--VI of FIG. 4.

FIG. 7 is a schematic view of a modified horizontal vessel according tothe invention.

FIG. 8 is a sectional view taken along the line VIII--VIII of FIG. 7.

FIG. 9 is a graph showing the results of a typical electrophoresismethod according to the invention.

FIG. 10 is a graph showing the results of a typical isoelectric focusingmethod according to the invention.

According to the invention, there is provided a hollow, elongated,horizontal, electrophoresis vessel which has electrophoresis electrodestherein adjacent the opposite ends thereof. An unobstructed zone isprovided in the interior of the electrophoresis vessel. The unobstructedzone is located at the bottom of the vessel when the vessel ispositioned in its normal position for electrophoresis. There also isprovided a multiplicity of upright, generally parallel, horizontallyspaced-apart partitions within the vessel defining a multiplicity ofseparate, isolated compartments which are located above the unobstructedzone in which the electrophoresis is carried out.

In the method according to the invention, a quantity of the startingspecies carried in a liquid electrophoresis medium is deposited into theunobstructed zone along the bottom of the electrophoresis vessel so asto form a liquid layer therein. This liquid layer remains substantiallystationary during the electrophoresis operation. Further, the liquidlayer is in contact with the electrophoresis electrodes. Anelectrophoresis potential difference is applied across the electrodeswhereby to cause the components of the starting species to migrateelectrophoretically in a horizontal direction in the layer whereby thestarting species becomes separated into fractions which are horizontallydisplaced from one another in a direction lengthwise of the vessel,owing to their different electrophoretic mobilities when electrophoresisis being carried out, or owing to their different isoelectric pointswhen isoelectric focusing is being carried out. After completion of theelectrophoresis operation, application of the electrophoresis potentialis discontinued, and then the vessel is turned through an arc about itshorizontal longitudinal axis so as to position the partitions lowermostwhereby to cause the liquid layer to flow into the compartments in theelectrophoresis vessel. Thereby, the respective fractions are separatelyreceived in separate isolated compartments in the electrophoresisvessel. The separated fractions can then be individually removed fromthe respective compartments and placed in different containers forfurther purification and/or analysis. Thus, from the starting species,there are recovered a multitude of different fractions containingdifferent components of the starting species and wherein the respectivefractions are more closely homogeneous than the starting species.

It is preferred that the unobstructed zone in the electrophoresis vesselcontains a lower substrate layer of liquid or solid, flowable a materialwhich is inert in the electrophoresis operation and which rests on thebottom of the vessel. The layer of the starting species andelectrophoresis carrier medium is deposited on the substrate layer,whereby to form an upper layer lying on top of the substrate layer. Inthis way, the starting species, in effect, floats above the substratelayer whereby to reduce sliding friction effects. It is preferred to usea lower substrate layer which is a liquid, such as a sucrose solution,in order to minimize friction. However, it is also within the scope ofthe broader aspects of the present invention to use a lower substratelayer which is a granulated gel of Sephadex (Trademark) or Bio-Gel(Trademark) material, both of which are well-known materials used in theelectrophoresis art.

The use of a lower flowable substrate layer of inert material is highlyadvantageous when the starting species is comprised of substances whichare sensitive to sliding friction, such as live cells. For example, if alayer containing a starting species comprised of lymphocytes were to beplaced directly on the bottom of the electrophoresis vessel, damage tothe lymphocytes could occur because of the movement of the lymphocytesalong and in contact with the bottom wall. In contrast, when thestarting species is placed as an upper layer on top of a lower flowablesubstrate layer, as described above, the starting species does notdirectly contact the bottom wall of the electrophoresis vessel and thereis much reduced opportunity for the occurrence of damage to the startingspecies.

Thus, the preferred embodiment of the present invention provides atwo-phase electrophoresis system wherein the starting species is presentin the upper phase of that system. That two-phase system can be asolution-solution system when the lower substrate layer is a liquid orit can be a solution-flowable solid system when the lower phasecomprises gel particles. Not only is the opportunity for damage to thestarting species reduced, but also the speed of separation of thefractions is improved by this two-phase technique. It is to be noted,moreover, that the method of the present invention does not involve thecreation of a density gradient within the upper layer, but rather, theelectrophoresis is carried out with the starting species dissolved orsuspended in a liquid carrier medium.

In carrying out a strictly electrophoresis separation under asubstantially constant pH, the liquid electrophoresis carrier medium isan electrically conductive liquid. Further, when the starting speciescomprises living things, such as lymphocytes, the electrophoresis mediumis also isotonic. On the other hand, when the starting species comprisesnon-living things, the electrophoresis carrier medium need be onlyelectrically conductive.

Further, with reference to strictly isoelectric focusing, theelectrophoresis medium comprises a carrier ampholyte liquid, such asAmpholine (trademark) liquids, which are commonly used in isoelectricfocusing.

Suitable liquid mediums for electrophoresis separations and isoelectricfocusing separations are well known to workers skilled in the art.Applicant's invention does not pertain to any discovery relating to suchmediums.

Referring to FIGS. 1 through 6 of the drawings, there is shown ahorizontal electrophoresis vessel which is useful for carrying out themethods described above. The horizontal electrophoresis vessel is ahollow, hexagonal, elongated housing 10 defined by six side wallportions 11, 12, 13, 14, 15 and 16 which are arranged to define a hollowhexagonal tube. The opposite ends of the hexagonal tube are closed bytwo hexagonal end walls 17 and 18. The side wall portion 11 is hingedlyconnected along one longitudinal edge thereof to the adjacent side wallportion 12 by hinges 19. The side wall portion 11 has a handle 21attached thereto so that the side wall portion 11 can be moved betweenopen and closed positions for access to the interior of the housing 10.

The housing 10 is made of electrically insulating material. It ispreferred that one or more of the wall portions 11-16 and the end walls17 and 18 are transparent to permit visual inspection of the contents ofthe housing 10 during the electrophoresis operation. In the illustratedpreferred embodiment of the invention, all of the wall portions 11-16and the end walls 17 and 18 are made of transparent synthetic resinwhich is suitable for use with the materials and under the conditionsemployed in electrophoresis operations. Moldable, transparent, acrylicresins, such as polymethyl methacrylate, are highly satisfactorysynthetic resins for this purpose.

A plurality of upright, parallel, horizontally spaced-apart partitions22 are mounted inside the housing 10. The partitions 22 are planar,trapezoidal plates which extend in planes perpendicular to thehorizontal longitudinal axis of the housing 10. The partitions extendfrom one side wall portion 12 along the side wall portion 13 to the sidewall portion 14 of the housing. The side edges and outer edge of thepartitions 22 are united to the inner surfaces of the side wall portions12, 13 and 14, whereby the partitions divide the zone between the sidewall portions 12, 13 and 14 into separate compartments 23 which areisolated from each other. The partitions 22 occupy less than one-half ofthe total cross-sectional area of the interior of the housing 10 and theinner edges 24 of the partitions are laterally outwardly spaced from thejuncture of wall portions 11 and 12 and from the juncture of wallportions 14 and 15. The space above the wall portion 15 up to the inneredges 24 of the partitions defines an unobstructed zone 25 in which theelectrophoresis is performed. Thus, when the starting species and theelectrophoresis liquid is placed in the housing 10 so as to form a layeron the bottom wall portion 15 thereof, as shown in FIG. 3, the level ofthe upper surface of the sample species is not higher than the lower endof the compartments 23, whereby the components of the starting speciesare capable of free lengthwise horizontal flow in the housing during theelectrophoresis operation without hindrance from the partitions 22.

The partitons 22 are made of electrical insulating material and,advantageously, they are made of the same synthetic resin as the housing10.

It will be appreciated that the housing 10 can be built up, for example,by adhesively securing together wall sections 11-16 together along theiradjacent edges. For example, the wall sections 12, 13 and 14 can beassembled to each other, the partitions 22 can then be mounted therein,following which the remaining wall sections 15, 16 and 11 can beattached thereto in order to complete the formation of the housing 10having the partitions 22 therein. The end walls 17 and 18 can besimilarly secured to the ends of the wall sections 12-16.

Electrodes 26 and 27 suitable for electrophoresis purposes are mountedon the end walls 17 and 18, respectively. The electrodes 26 and 27 arewire-like conductors made, for example, of platinum, and they arelocated close to and they extend substantially parallel to the interiorsurfaces of the end walls 17 and 18. The electrodes have lower legswhich extend into zone 25 and close to the wall portion 15 so that theycontact the electrophoresis liquid during the electrophoresis operation.The shapes of the electrodes 25 and 26 are not critical and they arehere shown as conforming roughly to the shape of the housing 10.

Electrical connectors 28 and 29 are mounted on the end walls 17 and 18at any suitable locations thereon. Here the connectors 28 and 29 arepositioned close to the juncture of the wall portions 11 and 12 andlaterally spaced from the inner edges 24 of the partitions 22. Theelectrodes 26 and 27 are connected to the connectors 28 and 29. Theconnectors 28 and 29 are adapted to be connected to the terminals of anysuitable electrophoresis power source so that one of the electrodesserves as the cathode and the other of the electrodes serves as theanode during electrophoresis.

The apparatus can be used to perform either electrophoresis orisoelectric focusing of the starting species in free solution. In bothmethods, the housing 10 is positioned horizontally with the wall portion15 being located lowermost so that the electrophoresis liquid rests onthe wall portion 15, as shown in FIG. 3. The electrophoresis potentialsource connected to the electrodes 26 and 27 applies an appropriateelectrophoresis potential and current for the appropriate period of timeeffective to separate the starting species into fractions. During suchelectrophoresis, the electrophorsis liquid, as a whole, remainsstationary on the wall portion 15 when the apparatus is placed on alevel horizontal surface, but the different molecules or bioparticles ofthe sample species will migrate in a lengthwise direction in the housingdifferent horizontal distances because of electrophoretic effects sothat the starting species becomes separated into fractions which arehorizontally displaced and are laterally aligned with different ones ofthe compartments 23 or at least different groups of adjacentcompartments. When electrophoresis is completed, the electrophoresispotential source is turned off. The housing 10 is then carefully turnedabout its horizontal longitudinal axis so as to rest on wall 14 and thenon wall 13, as shown in FIG. 4. The liquid thereby enters into therespective compartments 23 whereby the liquid becomes subdivided intoseparate electrophoresed fractions. This turning operation is carriedout as carefully as possible so as to minimize cross-contamination ofthe fractions due to unwanted mixing of horizontally adjacent fractions.The thus-separated fractions can be removed from the respectivecompartments and placed in separate receptacles, such as test tubes, forthe purposes of further separation and/or analysis.

The movable wall section 11 makes it possible to insert theelectrophoresis liquid into the vessel 10 and also to remove theseparated fractions from the vessel. During the electrophoresisoperation, the wall section 11 will be in the closed position in orderto prevent contamination of the contents of the vessel.

The location of the position at which the starting species is placed inthe vessel 10 prior to starting electrophoresis is not critical. Becauseit is desired to carry out the separation as rapidly as possible, theoperator will normally place the starting species at a position, in thelengthwise direction of the vessel 10, at which he expects theseparation to be completed most rapidly without cross-contamination ofthe fractions. This will depend on the specific components of thestarting species,, that is, whether they have a net positive or a netnegative charge in the case of electrophoresis, or the character oftheir isoelectric points in the case of isoelectric focusing. Normally,the operator will have had some experience which will lead him tobelieve that inserting the starting species at a particular locationwill give the best results. However, even if the place of insertion ofthe starting species is not the optimum location, still theelectrophoresis separation can be achieved, although possibly a longertime will be required. In the case of a starting species whosecharacteristics are not known, it will normally be placed atapproximately the longitudinal midpoint of the housing 10.

The number of compartments 23 in the vessel 10 can be selected to meetthe requirements of the particular separation desired to be effectedtherein. Although the number of compartments is not critical, it isgenerally useful in electrophoresis separations to use vesselscontaining from 10 to about 50 compartments.

MODIFICATION

Referring to FIGS. 7 and 8, there is illustrated a modifiedelectrophoresis vessel 10A which is supported for rotation by the axles31. The modified electrophoresis vessel is of circular cross section.The electrophoresis vessel 10A has a door section 11A to provide accessto the interior of the vessel and it also has a series of partitions 22Adisposed above the bottom portion of the vessel to provide compartments,like the compartments described above with respect to the principalembodiment of the invention. As shown, the partitions are in the form ofsegments of a circle and they occupy a relatively large proportion ofthe cross-sectional area of the central circular opening in the housing10A. The method of operation of the modified embodiment of the inventionwill be the same as described above with respect to FIGS. 1 through 6.

EXAMPLE 1

Heparinized venous blood was diluted twice with phosphate bufferedsaline (PBS) at pH 7.4 and was layered over Ficoll-Hypaque (Lymphoprep,Nyegaard Scientific) solution and centrifuged at 400×g for 20 minutes toobtain a lymphocyte-rich suspension. The suspension of the lymphocyteswas washed once with PBS and the concentration of the cells was adjustedto approximately 10⁹ per ml. Then, 1×10⁸ of the thus-isolatedlymphocytes in 2 ml of Hank's balanced salt solution were carefullyplaced on a layer of 25 ml of Ficoll-Hypaque solution in the midsectionof the electrophoresis vessel 10. In this position, the position of thevessel 10 was as shown in FIG. 3. The lymphocytes were subjected toelectrophoresis at 30 V, 25 ma for 3 hours, at room temperature.Electrophoresis was discontinued and then the vessel 10 was turned fromthe FIG. 3 to the FIG. 4 position, i.e., a counterclockwise turningthrough an arc of 120° whereby the separated fractions were collected inthe compartments. A reasonable separation of cell sub-populations wasobtained, as shown in FIG. 9. The average viability of the separatedcells was greater than 80%.

EXAMPLE 2

For electrofocusing of cells, 25 ml of Lymphoprep was mixed with 2 ml ofAmpholine (Trademark) (pH 5-7) over which 10⁸ lymphocytes in 0.2 mlHank's balanced salt solution were applied at the midsection of theelectrophoresis vessel positioned as shown in FIG. 3. The mixture ofcells and Lymphoprep was electrofocused at 25 V D.C. 20-25 ma for 4hours at room temperature. At the end of the electrophoresis, the vesselwas rotated to the FIG. 4 position in order to collect the separatedfractions. Each fraction was then transferred to a test tube and wascentrifuged. The results of this electrofocusing operation are shown inFIG. 10.

Although particular preferred embodiments of the invention have beendescribed above, the invention contemplates such changes ormodifications as lie within the scope of the appended claims.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method for separatinga starting species into separate fractions by electrophoresis orisoelectric focusing, in an elongated, horizontal vessel havinghorizontally spaced-apart electrodes therein, said vessel having anunobstructed zone along its bottom and having a multiplicity of upright,generally parallel, horizontally spaced-apart partitions defining amultiplicity of separate compartments above said zone, which comprisesthe steps of: depositing a quantity of said starting species carried ina liquid electrophoresis or isoelectric focusing medium in said zone toform a stationary liquid layer in said zone which layer is in contactwith said electrodes; then applying an electrophoresis or isoelectricfocusing potential across said electrodes for a period of time effectiveto cause said species to migrate electrophoretically in a horizontaldirection in said layer to separate said species into fractions whichare horizontally displaced from one another in a direction lengthwise ofsaid vessel; then turning said vessel about its horizontal longitudinalaxis whereby to cause said layer to flow into said compartments so thatthe respective fractions become separated from each other and arelocated in different ones of said compartments.
 2. A method according toclaim 1 in which said zone contains a lower flowable substrate layer ofinert material resting on the bottom of said vessel, said layer of saidstarting species and electrophoresis liquid being deposited to form anupper layer lying on top of said substrate layer so that said startingspecies floats above said substrate layer.
 3. A method according toclaim 2 in which said lower substrate layer is a liquid.
 4. A methodaccording to claim 2 in which said starting species is blood lymphocytesand said electrophoresis medium is an isotonic, electrically conductiveliquid.
 5. A method according to claim 1 or claim 2 in which saidstarting species is proteins and said isoelectric focusing medium is acarrier ampholyte liquid.